title: Gossypol acetic acid induces apoptosis in RAW264.7 cells via a caspase-dependent mitochondrial signaling pathway.
abstract: To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 μmol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (ΔΨm) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of ΔΨm in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.
authors.affiliation: College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China.
authors.forename: Sijun
authors.initials: S
authors.lastname: Deng
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.forename: Jine
authors.initials: J
authors.lastname: Yi
authors.forename: Yin
authors.initials: Y
authors.lastname: Lu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wei
authors.forename: Chengzhi
authors.initials: C
authors.lastname: Guo
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Liyun
authors.initials: L
authors.lastname: Yuan
authors.forename: Zuping
authors.initials: Z
authors.lastname: He
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006072:Gossypol / Q000031:analogs & derivatives* / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D051379:Mice; D008807:Mice, Inbred BALB C; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D017382:Reactive Oxygen Species; D006072:Gossypol; C028178:gossypol acetic acid
keywords: apoptosis; gossypol acetic acid; mitochondrial signaling pathway